Novel mechanisms of modulating GPCR function may result in improved therapeutics for treating many diseases including schizophrenia and depression. Studies performed during the previous funding cycle of this grant revealed two unexpected effects of drugs on h5-HT6 and h5-HT7 receptor activity. 1. Two groups of inverse agonists have been discovered: "high affinity/high potency" inverse agonists with potencies that are predicted from their affinities for the receptor, and "high affinity/low potency" inverse agonists with potencies that are far lower than predicted from their binding affinities for the receptor. 2.The second unexpected result is that risperidone (a widely prescribed antipsychotic drug), 9-OH-risperidone (the active metabolite of risperidone), and methiothepin produce a rapid and potent inactivation of the native h5-HT7 receptor (inactivating antagonists). Therefore, this proposal involves two specific aims: 1) determine the mechanism of action of high potency vs. low potency inverse agonists at CAM h5-HT6 and 5-HT7 receptors;and 2) determine the mechanism(s) of action that results in risperidone's rapid and potent inactivation of the native h5-HT7 receptor. Specific aim"1 will be approached in three ways: a) detailed pharmacological analysis of the actions of inverse agonists at the CAM h5-HT6 and h5-HT7 receptors to determine if an allosteric mechanism may be involved;b) monitor the effects of inverse agonists on CAM h5-HT6 and h5- HT7 receptor internalization and beta-arrestin translocation;c) monitor the effects of inverse agonists on CAM h5-HT6 and h5-HT7 receptor associated MARK activity. Specific aim 2 will be approached in three ways: a) determine if inactivating antagonists interact irreversibly with the native h-5HT7 receptor in intact cell preparations;b) monitor the effects of inactivating antagonists on native h5-HT7 receptor internalization and beta-arrestin translocation, and c) monitor the effects of inactivating antagonist treatment on MARK activity in cells expressing native h5-HT7 receptors. The results from these studies may reveal novel mechanisms for modulating the functional state of h5-HT6 and h5-HT7 receptors, which may be applicable to many other GPCR. Dysfunctions of these modulating mechanisms may underly the psychopathology of various mental diseases. These studies may lead to the development of novel therapeutics for brain dysfunctions, including schizophrenia and depression.